Effects of acupuncture treatment for myasthenia gravis: A systematic review and meta-analysis

Background Randomized controlled trials (RCTs) of acupuncture for myasthenia gravis (MG) were searched and the efficacy of acupuncture in the treatment of MG was evaluated by meta-analysis. Methods We searched for RCTs in six main electronic databases, and collected RCTs of acupuncture treatment for MG from database creation to 28 February 2023. The main outcome was the effective rate and the secondary outcome was the Traditional Chinese Medicine (TCM) relative clinical score, absolute clinical score (ACS) of MG, Quantitive myasthenia gravis score (QMG), quality of life, and adverse events. Odds ratios (ORs) and weighted mean differences (WMD) and 95% confidence intervals (CI) were used to assess pooled effect estimates using Review Manager software. Results A total of 14 RCTs were included. Meta-analysis showed that the effective rate in the acupuncture group was significantly improved compared with conventional Western medicine alone [OR = 4.28, 95% CI (2.95, 6, 22), P<0.005]. The pooled WMDs revealed that TCM relative clinical score [WMD = -2.22, 95% CI = (-2.53, -1.90), P<0.005], ACS of MG [WMD = -3.14, 95% CI = (-3.67, -2.62), P<0.005], and QMG [WMD = -0.88, 95% CI = (-1.46, -0.29), P<0.005] in the acupuncture group was lower than the control group. Adverse reactions related to acupuncture and quality of life were less mentioned among included RCTs. Conclusion This meta-analysis demonstrated that acupuncture as an auxiliary may play a positive role in treating MG. It can improve the effective rate of treatment, and reduce TCM relative clinical score, ACS of MG, and QMG. However, the quality of included studies was generally low and caution should be exercised when considering this treatment option. In the future, more rigorous study designs and high-quality RCTs are needed to verify the efficacy of acupuncture in the treatment of MG, because the results of high-quality RCTs are more reliable and accurate.


Introduction
Myasthenia gravis (MG) is an acquired autoimmune disease caused by neuromuscular postsynaptic membrane damage and abnormal transmission function at the neuromuscular junction, resulting in muscle weakness [1,2].The pathogenesis of MG is mainly related to acetylcholine receptor mediated, cellular immune dependence and complement participation [3,4].The clinical manifestations of MG are fluctuating muscle weakness and fatigue of various muscle groups, and dyspnea may occur in severe cases [5].The age of onset ranged from young to late adulthood, with the peak incidence in young adult women and elderly men [6].The incidence is estimated at 10 to 25 per 100,000 and the worldwide prevalence at 700,000 [6,7].
At present, conventional treatments of MG include: acetylcholinesterase inhibitors (AchEI), immunosuppressants, plasma exchange, thymectomy, etc [7,8].Pyridostigmine is one of the common AchEI for the treatment of MG.Its mechanism is to inhibit the activity of cholinesterase (AChE) by binding to AChE, which increases the concentration of acetylcholine (ACh) in the neuromuscular junction, thereby stimulating ACh receptor to improve the symptoms of muscle weakness [9].Prednisone and tacrolimus are commonly used immunosuppressants.Studies have demonstrated that early use of immunosuppressants can improve the prognosis of MG patients, such as reducing the recurrence rate and improving the quality of life of patients [10].However, these costly treatments cannot completely cure MG, and most patients are easy to appeared adverse reactions of drugs, such as diarrhea, nausea, vomiting, hydrostomia, and even muscle twitching and dependence [7,8].Traditional Chinese medicine (TCM) including traditional Chinese medicine decoction, Chinese patent medicine, acupuncture and other therapies have certain advantages in improving the efficiency of disease treatment, reducing the recurrence rate, reducing side effects and improving the condition [11].For example, Shengyang Jutuo decoction, Buzhong Yiqi decoction, etc. are commonly used Chinese medicine decoction which alleviates symptoms by improving patient's autoimmune status in China.As an important part of Chinese medicine, acupuncture has been commonly practiced treat various diseases in clinical practice in China [11].As a non-pharmacological treatment, acupuncture rarely causes adverse effects such as gastrointestinal discomfort, liver function damage, and kidney function damage because it does not involve pharmacological metabolism and other effects.Acupuncture can dredge the meridians, regulate "Yin" and "Yang", stimulate local nerves quickly and directly through physical action, and improve the local immune microenvironment, which has been confirmed in many animal experiments and clinical observations [12].There are many types of acupuncture, such as fire acupuncture, electric acupuncture, manual acupuncture, warm acupuncture, water acupuncture, etc [13,14].These methods have less expensive and few side effects.
With the deepening of TCM treatment of MG, many studies have shown that acupuncture combined with drugs has therapeutic advantages in the treatment of MG [15].So far, systematic reviews of acupuncture for MG are relatively scant, whereas most of the existing randomized control trials (RCTs) are limited by the small sample size and study design flaws, which may bring about controversial results and cannot provide adequate evidence for further clinical applications.Therefore, we conducted a systematic review and meta-analysis of published 12 RCTs using the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines, aiming to evaluate the efficacy and safety of acupuncture in the treatment of MG, and provided reliable evidence-based medical evidence for the precise treatment of MG [16].

Material and methods
This meta-analysis (registration No. CRD42023402885) focused on RCTs involving acupuncture interventions on myasthenia gravis, which abided by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement [17].

Eligibility criteria
Articles meeting the following criteria were included: (1) population: patients were diagnosed as myasthenia gravis according to WHO diagnostic criteria, which were based on typical clinical features of MG and any of the following three conditions, including neostigmine test, electrophysiological features, and serum anti-ACh antibody test; (2) interventions: patients in the experimental group received acupuncture therapy, including manual acupuncture, electronic acupuncture, and scalp acupuncture, or acupuncture therapy with medicine therapy; (3) control: patients in the control group received medicine therapy, placebo treatment such as sham acupuncture, or no treatment; (4) outcome measures: the main outcome focused on the effective rate.The clinical efficacy of MG was considered meeting one of the following conditions as effective: 1) Traditional Chinese medicine (TCM) relative clinical score decreased by more than 25% compared with the previous; 2) Improvement of symptoms.The effective rate was regarded as a dichotomous measure (effective or ineffective).[18] The secondary outcome measures included Traditional Chinese medicine (TCM) relative clinical score, absolute clinical score(ACS) of myasthenia gravis, Quantitive myasthenia gravis score (QMG), quality of life, and adverse events; (5) RCTs regardless of publication language were Chinese and English.The following were excluded from the analysis: non-RCTs, uncontrolled trials, protocols for RCTs and inappropriate intervention studies, such as comparisons between two groups focusing on different acupuncture methods or different acupuncture points.

Search strategy
From database creation to 28 February 2023, we searched the following six electronic databases: Cochrane Central Register of Controlled Trials (Central), PubMed, Embase, China National Knowledge Infrastructure (CNKI), China Biomedical Literature Database (CBM) and Wanfang Database.We manually searched the relevant literature to identify other potentially eligible studies.The keywords for literature retrieval were "acupuncture," "acupuncture therapy," "electroacupuncture," "acupuncture treatment," "myasthenia gravis," and "randomized controlled trial."The search terms and search strategy are presented in S1 Table.

Data collection
Microsoft Excel was used to compile electronic data extraction forms and manage information extracted from eligible articles, including country, year of publication, sample size unlimited, mean age of patients, interventions in the experimental group, interventions in the control group, and outcomes (include effective rate, TCM relative clinical score, ACS, QMG, quality of life, and adverse events).Investigators extracted and verified all data to ensure accuracy.
Corresponding authors were contacted and asked to clarify any ambiguities and, if possible, submit missing information by phone or email.

Quality assessment
Two reviewers independently assessed the quality of the included trials using the Cochrane Collaboration tool to assess the risk of bias (ROB) which provides a rigorous independent assessment of different terms of study quality.The Cochrane Collaboration tool assesses seven important sources of bias including random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting, and other.The reviewer' s judgment for each item including "low risk", "high risk" or "unclear"of bias.Any disagreements were settled by discussion with another reviewer.

Data synthesis and statistical methods
Data were analyzed using Cochrane Collaboration Meta-analysis software (Review Manager 5.4).Dichotomous data, such as effective rate was expressed by odds ratio (OR) or relative risk (RR) and their 95% confidence interval (CI).Continuous data such as TCM relative clinical score, ACS of MG, quantitive myasthenia gravis score were expressed by weighted mean difference (WMD) and its 95% CI, and P<0.05 was considered statistically significant.The heterogeneity test was carried out on the included literature.If P �0.1,I 2 � 50%, it indicated the existence of heterogeneity, and the random effect model was used for heterogeneity analysis.Statistical heterogeneity or less heterogeneity, using a fixed-effects model.The results of metaanalysis were expressed in forest plots, and funnel plots were used to evaluate publication bias if included articles more than 10.

Study selection
A comprehensive search of the database from database creation to 28 February 2023 retrieved a total of 891 studies that met the relevant criteria, and 352 duplicates were removed using the literature manager.After reading the titles and abstracts, 471 irrelevant studies were excluded, and 67 literature were selected for full-text reading.54 of the 67 literature were excluded, and the detailed reasons are shown in the flow chart.Fourteen RCTs were finally included for meta-analysis [18][19][20][21][22][23][24][25][26][27][28][29][30][31].(Fig 1).

Study characteristics
Detailed characteristics of the included studies including publication year, first author, country, sample size, age, treatment and control group intervention, and treatment period are described in Table 1.The selection of acupoints is shown in Table 2.All studies were conducted and published in China between 2010 and 2022, and a total of 1009 patients with MG were enrolled.With sample capacity ranging from 34 to 154 and therapy duration ranging from 4 weeks to 24 weeks.None of the studies used sham acupuncture as a control.The control group was mainly prednisone or pyridostigmine bromide.Four studies reported a followup of 1 to 3 months, while none of the others reported follow-up.Thirteen trials reported effective rate, 4 trials reported TCM relative clinical score, five trials evaluated ACS of MG, and two RCTs reported QMG.

Quality assessment of included studies
Ten studies described in detail the method of generating random sequences, including random number tables, computer-generated random numbers.The remaining four studies did not explain the process.3 of the 14 studies described the process of allocation concealment.In the remaining studies, the allocation concealment and blinding processes were not clearly described.Most of the included RCTs were considered to be at "high risk" for performance bias and detection bias.Because 2 RCTs had missing patients, we conclude that both trials were at "high risk" for incomplete outcome data.Moreover, 12 RCTs had a sample size of less than 100, and eight RCTs did not report follow-up.The overall quality of the RCTs was not high, as most RCTs were rated as "unclear" and "high risk".Clear details of the methodological quality assessment are provided in Figs 2 and 3.

Effective rate. 13 included studies involving 952 patients reported effective rate.
There was no statistical heterogeneity among the studies (P = 0.63, I 2 = 0%), and the fixed effect model was used for analysis (Fig 4).This results of Meta-analysis showed that the effective rate of the experimental group was higher than that of the control group [OR = 4.28, 95% CI = (2.95,6.22) ], and the difference was statistically significant (P < 0.001).

Traditional Chinese Medicine (TCM) relative clinical score.
A total of four RCTs were included with 247 patients assessed TCM relative clinical score.The heterogeneity among the studies was mild (P = 0.78, I 2 = 0%), and the fixed effect model was used.The results of meta-analysis showed that the TCM relative clinical score of the experimental group was lower than the control group [WMD = -2.22,95% CI (-2.53, -1.90) ], and the difference was statistically significant (P < 0.001) (Fig 5).

ACS of myasthenia gravis.
A total of five studies evaluated the ACS of myasthenia gravis.There was significant heterogeneity between studies (P = 0.004, I 2 = 61%), random effect model was used for analysis.The results of Meta-analysis revealed that the ACS of myasthenia gravis in the experimental group was significantly lower than the control group [WMD = -3.14, 95% CI = (-3.67,-2.62)], and the difference was statistically significant (P < 0.001), suggesting that the experimental group was superior to the control group in improving the clinical symptoms of myasthenia gravis (Fig 6).
3.4.5Quality of life and adverse events.Two RCTs involving 106 MG patients evaluated the quality of life.The SF-36 Questionnaire was used to investigate the quality of life of MG patients in one study.Another study used the Busch Modified Quality of Life Scale.The research results suggested that acupuncture as an auxiliary means has a positive effect in improving the quality of life.Only one study mentioned adverse events, that is, there were no obvious adverse events between the two groups.

Publication bias
The effective rate from more than 10 studies were assessed for publication bias, and publication bias assessments are presented as funnel plots (Fig 8).From the roughly symmetrical shapes of these funnel plots, no obvious publication bias was observed.

Discussion
In this meta-analysis on acupuncture treatment for MG, a total of 14 relevant literatures were included, and the included subjects were all patients who met the diagnostic criteria of relevant MG.A total of 1009 patients were enrolled in the analysis, including 507 patients in the experiment group and 502 patients in the control group.The main outcome indicator included in this meta-analysis was the effective rate.The secondary outcome indicator were TCM relative clinical score, ACS, QMG.The literature on adverse reactions and recurrence rates after treatment included in this study is less described, and the possibility of selective reporting by researchers cannot be ruled out.Considering the limited number of literature included in this meta-analysis, and the concept of acupuncture treatment is broadly, there are other clinical operation methods involving the concept of acupuncture treatment that have not been included, such as acupoint direct current stimulation, acupoint injection, auricular point sticking, auricular magnetic stimulation and other programs have not been reflected [32].This meta analysis results suggested that acupuncture or acupuncture combined drugs can improve  [33].In addition, modern studies have shown that acupuncture can promote the functional activity of acetylcholine [24].
The comprehensive demonstrated that acupuncture as auxiliary has a positive effect on treatment of MG.However, the effect of acupuncture on improving quality of life is unclear.Only 2 studies assessed quality of life and did not use the same scale.Most studies have focused on the effectiveness of acupuncture, and little attention has been paid to the side effects.There is uncertainty about the adverse events of acupuncture due to insufficient attention to adverse events in RCTs.
Through searching databases, Jin et al. found high-frequency acupoints for the treatment of MG were Zu-san-li (ST36), Yang-bai (GB14), Cuan-zhu (BL2), and Yu-yao.Zu-san-li (ST36)-Cuan-zhu (BL2) combination was preferred.The mechanisms of acupuncture in the treatment of MG are mainly as follows: (1) Acupuncture can reduce the expression of Forkhead Box Class O3a (FOXO3a) transcription factors in atrophic skeletal muscle tissue.By inhibiting the activation of FOXO3a, it can reduce the expression of Muscle Atrophy Fbox (MAFbx) and Muscle Ring Finger-1 (MuRF1) mRNA, thereby preventing the degradation of muscle proteins, increasing the cross-sectional area of muscle fibers, and improving the degree of skeletal muscle atrophy; [34] (2) Acupuncture can improve the level of mitochondrial fission and fusion.By increasing the expression of fusion protein Optic Atrophy 1 (Opa1) and fission protein Dynamin-related Protein 1 (Drp1), a new balance between mitochondrial fusion and fission is established.This balance can promote the synthesis and function of mitochondrial ATP, thereby enhancing muscle strength; [35] (3) Acupuncture can excite the cholinergic N receptor and activate the cholinergic anti-inflammatory pathway (CAP), thereby exerting an anti-inflammatory effect and delaying the progression of myasthenia.
The present study suggested that acupuncture may be beneficial for patients with MG and provides a new treatment for the clinical practice of MG.The advantages of traditional medicine and modern medicine can complement each other to help patients rebuild their immune function and reduce adverse reactions and recurrence rates.However, the sample size of RCTs included in this study was small and all were from China, and the effectiveness, safety, durability, and acceptability of acupuncture for MG hinder its widespread use in the treatment of MG.We look forward to higher quality RCTs, including placebo-controlled trials, larger sample quantity, more final indicators, and longer follow-up duration to evaluate clinical significance and safety.In addition, attention should be paid to adverse events in RCTs in the future.More animal acupuncture trials in the future are also needed to further validate and clarify the underlying mechanisms of acupuncture for MG.
This study has the following limitations: (1) In terms of literature quality, the quality of the included pieces of literature are generally low, all of which are in Chinese which may lead to a language bias and limit the generalizability of the findings, most of which not mentions blinding of outcome assessment use; (2) Intervention measures on the one hand, the drug dosage, treatment time, and follow-up time of individual studies are inconsistent, leading to differences in observation results and reducing the strength of evidence for conclusions; (3) In terms of outcome indicators, there are few literatures that include absolute clinical scores and TCM relative clinical score for MG.The included articles mentioned that adverse reactions and recurrence rates were less described, and selective reporting cannot be ruled out; (4) In terms of follow-up period, only four RCTs mentioned follow-up periods ranged 1 months to 3 months.In order to improve the evidence strength of the conclusion, more high-quality, largesample, multi-center clinical randomized controlled trials are needed to comprehensively report the improvement of clinical symptoms, adverse reactions, and immune function indicators of MG, so as to further to verify the efficacy and safety of acupuncture in the treatment of MG.

Conclusion
This meta-analysis of this study suggests that acupuncture is generally effective as an intervention for MG, providing evidence-based evidence for the use of acupuncture for MG.An integrated approach of acupuncture combined with conventional therapies may provide benefits for MG patients.At the same time, due to the small number of cases included in the literature, the random method of the original literature is not rigorous enough, and the quality of the literature after literature review is relatively low, which will lead to discrepancies between this meta and the literature.These results should be treated with caution.

Table 1 . Characteristics of included studies and patients.
④ T, intervention group; C, comparison group; tid, three times a day; qd, one time a day; ①, effective rate; ②, TCM relative clinical score, Traditional Chinese Medicine relative clinical score; ③,Quality of life; ④, ACS of myasthenia gravis, Absolute clinical score of myasthenia gravis; ⑤, QMG, Quantitive myasthenia gravis score.⑥,